Overexpression of CBS/H2S inhibits proliferation and metastasis of colon cancer cells through downregulation of CD44

Background The role of hydrogen sulfide (H2S) in cancer biology is controversial, including colorectal cancer. The bell-shaped effect of H2S refers to pro-cancer action at lower doses and anti-cancer effect at higher concentrations. We hypothesized that overexpression of cystathionine-beta-synthase (CBS)/H2S exerts an inhibitory effect on colon cancer cell proliferation and metastasis. Methods Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), clone-formation and sphere formation assay. Cell migration was evaluated by transwell migration assay. Intracellular H2S was detected by H2S probe. Chromatin immunoprecipitation (ChIP) analysis was carried out to examine DNA–protein interaction. Cell experiments also included western blotting, flow cytometry, immunohistochemistry (IHC) and immunofluorescence analysis. We further conducted in vivo experiments to confirm our conclusions. Results Overexpression of CBS and exogenous H2S inhibited colon cancer cell proliferation and migration in vitro. In addition, overexpression of CBS attenuated tumor growth and liver metastasis in vivo. Furthermore, CD44 and the transcription factor SP-1 was probably involved in the inhibitory effect of CBS/H2S axis on colon cancer cells. Conclusions Overexpression of CBS and exogenous provision of H2S inhibited colon cancer cell proliferation and migration both in vivo and in vitro. Molecular mechanisms might involve the participation of CD44 and the transcription factor SP-1. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-022-02512-2.


Background
Colorectal cancer (CRC) is a life-threatening disease with the second-highest mortality of all human malignancies worldwide [1]. It is also the second-most common cancer in China, accounting for 12.2% of new cancer cases in 2020 [1]. However, the biological and molecular mechanisms of CRC are still elusive and liver metastases remain a major contributor of cancer related mortality in CRC.
Hydrogen sulfide (H 2 S), the third 'gasotransmitter' in addition to nitric oxide (NO) and carbon monoxide (CO), is synthesized mainly from L-cysteine through three pivotal enzymes: cystathionine-beta-synthase (CBS), cystathionine-gamma-lyase (CSE) and 3-mercapto-pyruvate sulfurtransferase (MPST) [2][3][4]. Endogenous H 2 S is widely involved in the delicate regulation of various physiological conditions, including nervous [5], cardiovascular [6], renal [7], gastrointestinal [8], reproductive [9] and respiratory [10] systems. Colon epithelium is constantly exposed to high levels of H 2 S derived from gut microbial metabolism [11]. Increased sulfide oxidation pathway, mainly composed of sulfide quinone oxidoreductase (SQR), thiosulfate-dithiol sulfurtransferase (TST) and ethylmalonic encephalopathy protein 1 (ETHE1), has been involved in detoxifying H 2 S in colon epithelial cells [11]. Previous studies including our work have validated the upregulation of CBS/H 2 S in CRC tumor specimens compared with patient-matched nonmalignant tissues [12]. Endogenous H 2 S was involved in the metabolic reprogramming and tumor associated angiogenesis in the CRC tumor microenvironment, contributing to the chemo-resistance phenotype. However, a plethora of studies have focused on the inhibitory effect of high levels of exogenous H 2 S on the proliferation and metastasis of cancer cells and several H 2 S donors have been developed. The bell-shaped model of the physiological role of H 2 S provided a convincing explanation to this paradox, which refers to pro-cancer action at lower doses of H 2 S and potential anti-cancer effect at higher levels [13]. Considering the dichotomous effect of H 2 S and the development of new era H 2 S donors, our study set out to investigate the declining part of the bell and hypothesize that overexpression of CBS/H 2 S might exhibit an anticancer activity on CRC cells.
CD44, a transmembrane glycoprotein and an important biomarker of cancer stem cells (CSCs) [14][15][16], is essential to many tumor cell activities, including proliferation and metastasis. CD44 has several variant isoforms generated through alternative mRNA splicing and these isoforms are reported to be associated with higher metastatic potential and poorer prognosis in various cancers, including CRC [17][18][19][20][21]. To our knowledge, there are no existing studies linking gasotransmitter H 2 S with cell surface molecule CD44 in any way.
Our results show that overexpression of CBS/H 2 S indeed exerts an inhibitory effect on the proliferation and migration of colon cancer cells both in vitro and in vivo. Mechanistically, CBS overexpression inhibits the expression of CD44, probably via attenuating the activation and nuclear translocation of the transcription factor SP-1.

Gene editing
KO-(sh-CBS) was performed using lentiviruses with short hairpin RNA (shRNA) targeting CBS mRNA sequence (Sigma, USA). Lentiviruses with shRNA of scrambled sequence served as negative control (Sigma, USA). Caco-2 cells were infected at a MOI of 30 with 10 μg/ml of polybrene, and were then selected with 6 μg /ml puromycin for 7 days. Similarly, overexpression of CBS for HT-29 cell line was achieved through transfection with lentiviruses containing h-CBS sequence (Hanbio Biotechnology Co., Ltd., China) at a MOI of 30 with 10 μg/ml of polybrene followed by screening with 4 μg / ml puromycin for 7 days.
Stable knockout of CBS (KO-CBS) for HT-29 cell line was conducted using CRISPR/Cas9 system. Gene-specific sgRNA was designed to target CBS coding regions as shown below: CTG ATG AGA TCC TGC AGC AG. We first phosphorylated, annealed, and cloned the guide oligonucleotide into the BsmBI site of the pHBLV-U6-gRNA-EF1-CAS9-PURO vector (Hanbio Biotechnology Co., Ltd., China), and then verified the constructed vector by sequencing. Next, we transformed the transfer plasmid with the oligonucleotide into Escherichia coli strain DH5α bacteria, and used Plasmid DNA purification kit (Macherey-Nagel, Germany) to isolate the amplified plasmid from the bacteria. Transferable lenti-CAS-puro plasmid (Hanbio Biotechnology Co., Ltd., China), packaging plasmids psPAX2 (Hanbio Biotechnology Co., Ltd., China) and pMD2G (Hanbio Biotechnology Co., Ltd., China) were transfected into 293T cells to produce the lentivirus. Virus-containing supernatant was collected at 48 and 72 h post transfection and was used to infect Caco-2 cells. Sixteen hours after the infection, fresh medium containing 10 μg/ml puromycin was utilized to replace the lentivirus-containing medium. We collected the puromycin-resistant cells after 7 days of screening.
Finally, western blotting was used for all gene-edited cell lines to assess the silencing or overexpression efficiency.

Cell counting kit-8 (CCK-8) assay
CCK-8 assay was performed to measure the capacity of cell proliferation using CCK-8 kit (Sigma-Aldrich, USA). Cells were seeded in 96-well plates at a density of 4000 cells per well with or without GYY4137 supplement. After incubation, 10 μl CCK8 were added to the wells at different time. The absorbance was measured at 450 nm by microplate reader (Bio-Rad, Hercules, CA, USA).

Colony formation assay
Colony formation assay was used for cell proliferation analysis. Cells were seeded into 6-well plates at a density of 1000 cells per well for 14 days. Colonies were then fixed with 10% formaldehyde for 20 min and stained with 0.1% crystal violet for 10 min at room temperature. Colonies containing more than 50 cells were counted and the mean colony numbers were calculated. Each clone was plated in triplicate in each experiment.

Transwell migration assay
For migration assay, 2.5 × 10 5 cells suspended in 200 μl serum-free medium were added into the upper transwell chamber (8 mm, Corning Costar, USA), and 700 μl medium supplemented with 20% serum was placed in the lower chamber. Each transwell chamber contains a 6.5 mm diameter membrane with 8.0 μm pore size. After incubation, cells on the upper surface of membrane were removed gently with a cotton swab. Cells invading to the lower surface were fixed with methanol before staining with 0.1% crystal violet for 20 min at room temperature. The stained cells were counted in 5 randomly selected fields under a light microscope. Each clone was plated in triplicate in each experiment.

Real-time quantitative PCR (RT-qPCR)
Total RNA from xenografts and cell lines were isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Total RNA (1 mg) was eluted with RNase-free water and stored at -80 °C. RT-qPCR was performed using SYBR-green PCR Master Mix in a Fast Real-time PCR 7500 System (Applied Biosystems). The primers for CD44 were as follows: 5′-CCT TTG ATG GAC CAA TTA CCA TAA C-3′ (forward); 5′-TCA GGA TTC GTT CTG TAT TCT CCT T-3′ (reverse). GAPDH was used as the internal control. Fold change of CD44 was calculated by the 2 −ΔΔCt method.

Western blotting
Total protein was extracted using RIPA buffer and the extracts containing equal quantities of protein (30 μg) were electrophoresed in 10% polyacrylamide gel, transferred with PVDF membranes and blocked for 1 h (5% BSA in TBS-Tween 20 buffer) at room temperature. Incubations with primary antibodies to detect CD44, CBS, and β-actin (CST, USA) were followed by incubations with secondary antibodies conjugated with horseradish peroxidase (CST, USA). Blots were developed with ECL detection reagents (Millipore, USA). Images were collected utilizing Syngene GeneGenius gel imaging system (Syngene, UK) according to the manufacturer's instructions.

Immunohistochemistry (IHC) analysis
Xenografts from subcutaneous injection and liver samples were immediately fixed in 10% neutral buffered paraformaldehyde. After fixation, the tissue was dehydrated in a graded ethanol series and then embedded in paraffin. Each section (3-μm) of the paraffin-embedded tissue was mounted on a glass slide and either stained with hematoxylin and eosin (H&E) or processed for IHC. For the latter, each slide was completely deparaffinized by immersion in xylene twice for 10 min and rehydrated with water following incubation in graded ethanol (100, 90, 80, and 70%). The antigen retrieval procedure was carried out by microwaving the slides for 10 min in citrate buffer (pH 6.0; Biogenex, San Ramon, CA) followed by incubation in 3% H 2 O 2 in methanol to block endogenous tissue peroxidase activity. The sections were blocked with 1.5% goat serum for 1 h and incubated with CD44 or Ki-67 antibody overnight at 4 °C. Mouse anti-CD44 (CST, USA) and mouse anti-Ki-67 (CST, USA) antibody was used. After washing with PBS, slides were then incubated with a biotinylated secondary antibody for 30 min at room temperature. The antigen signal was amplified using the ABC method (Vectastain ABC kit, catalogue no. PK-6105, Vector Laboratories, Burlingame, CA). The antigen-antibody-avidin complex was detected using the chromogenic substrate 3,3-diaminobenzidine, which produced a dark brown color. Immunostained sections were counterstained with hematoxylin and examined by light microscopy. H-score was used for the semi-quantitive analysis of IHC results. The calculation of H-score was based on the intensity of staining (3, strong; 2, moderate; 1, weak; 0, none) and the proportions of positively stained tumour cells as previously described (H-score = % strong staining × 3 + % moderate staining × 2 + % weak staining × 1 + % no staining × 0) [22]. For each sample, five fields were randomly selected and the average H-score was calculated.

Immunofluorescence assay
For the immunofluorescence assay, after fixation with 4% paraformaldehyde for 10 min, PBS was used to gently wash the cells thrice. Then, the cells were immunostained with primary antibodies targeting SP-1 (CST, USA) at 4 °C overnight, and the secondary antibodies used were Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific, USA). Lastly, the cells were again washed with PBS and then mounted with ProLong Gold mounting medium with DAPI (Molecular Probes, USA). The sections were observed by confocal laser scanning microscopy (Zeiss LSM780, Carl Zeiss, Germany). Pearson's correlation coefficient was used for the semi-quantitive analysis of the colocalization values as previously described [23].

Fluorescent detection of intracellular H 2 S
To visualize intracellular H 2 S level, 2 × 10 4 cells were seeded in a glass-bottom 35 mm well (Corning, USA) and cultured overnight. After adding 10 µmol/L of H 2 S-specific near-infrared fluorescence probe to the culture medium and incubated for 30 min, the living cells were immediately sent for fluorescence imaging [24].

Flow cytometry
For surface marker detection, the cells were collected and resuspended at a density of 1 × 10 4 per test. After incubation for 30 min at room temperature with CD44-APC antibody (eBioscience, USA), the cells were washed twice with PBS and resuspended in 400 µL of PBS for flow cytometry using Calibur 2 (Beckman Coulter, USA). The results were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). The mean fluorescence intensity (MFI) ratio (sample ΔMFI (specific marker MFI − isotype control MFI)/control sample ΔMFI × 100) was calculated for all samples.

Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed according to the instructions of Agarose ChIP Kit (Thermo Fisher Scientific, USA). Antibody against SP-1 (CST, USA) was used to precipitate the DNA-protein complex and subsequently elute the DNA from the antibody. Primers specific for the CD44 promoter were 5′-CTC TTT CCA CTT GGA AGA TTC ACC A-3′ (forward) and 5′-TGG ATA TCC TGG GAG AGG AGCT-3′ (reverse). The immunoprecipitated DNA was amplified by real-time PCR using SYBR-green PCR Master Mix in a Fast Real-time PCR 7500 System (Applied Biosystems).

Xenograft model
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University First Hospital. Twelve male BALB/c nude mice at age of 6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.. Each mouse was subcutaneously inoculated with 1 × 10 6 of HT-29 cells in a volume of 200 μl PBS with or without CBS overexpression. After a palpable tumor was developed, the tumor length A and width B were measured twice a week using a caliper. The formula used for calculating the tumor volume was A × B2/2. Mice were humanely sacrificed by exposure to a fixed flow rate of CO 2 (30% chamber replacement rate) 34 days after inoculation. Subcutaneous tumor grafts were harvested and analyzed by western blotting and IHC analysis.

Intrasplenic injection model
Twelve 6-week-old male Balb/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.. To investigate the tumor metastasis in vivo, 1 × 10 6 of HT-29 cells containing stablyexpressed human CBS sequence or empty vector in a volume of 100 μl in PBS were injected into spleen subcapsular followed by spleen-resection after 5 min. After 8 weeks, the livers of nude mice were surgically removed after euthanasia with CO 2 (30% chamber replacement rate), fixed in 10% neutral buffered formalin, embedded in paraffin, and prepared into 3-μm sections for H&E staining and IHC analysis.

Statistical analysis
Data are expressed as means ± SD. All statistical analyses were undertaken using Prism for macOS software, version 8.4.1 (GraphPad Software, La Jolla, CA, USA). Student's t-test or the Mann-Whitney U-test were used for comparisons between two experimental groups with or without normal distribution, respectively. p-values < 0.05 were considered statistically significant.

Overexpression of CBS inhibits HT-29 cell proliferation, clone formation, sphere formation, and migration
In colorectal tumor tissue and cancer-derived cell lines, the expression of CBS is upregulated and closely related to tumor growth and carcinogenesis. At the same time, the dual-effect of several gasotransmitters, including H 2 S, CO and NO, has been reported. To investigate the biological function as well as therapeutic potentials of CBS/H 2 S axis, HT-29 cell line with stable overexpression of CBS was generated. The approximately twofold upregulation of CBS was confirmed by western blotting, and the increased production of H 2 S was verified by a fluorescent H 2 S probe through fluorescence analysis (Fig. 1A, B). To examine whether overexpression of CBS affects CRC cell proliferation, we performed a CCK-8 assay and the results showed that overexpression of CBS significantly inhibited HT-29 proliferation from 24 to 72 h (vs. control vector, *p < 0.05, **p < 0.01; Fig. 1C). Consistent with the cell viability analysis, a colony formation assay indicated that overexpression of CBS significantly reduced the number of HT-29 cell clones (**p < 0.01; Fig. 1D, E). Next, to further examine the effect of CBS overexpression on cell-renewal capacity, we conducted a sphere formation assay. As expected, CBS-overexpressing HT-29 cells formed smaller and less spheres than that of control cells (**p < 0.01; Fig. 1F, G). Finally, we explored whether CBS overexpression affected the migration of HT-29 cells using 24-h transwell assays. We observed that cell migration was significantly attenuated in CBSoverexpressing HT-29 cells (***p < 0.001; Fig. 1H, I). In general, these results suggest that CBS overexpression inhibits CRC cell proliferation, clone formation, sphere formation, and migration in vitro.

Exogenous H 2 S inhibits CRC cell proliferation and migration.
To further determine the inhibitory effect of CBS overexpression and the resulting increased rate of H 2 S production on CRC cells, we treated both HCT-116 and HT-29 cells with a slow-releasing H 2 S donor, GYY4137. We first visualized the elevated intracellular H 2 S concentration with the fluorescent H 2 S probe ( Fig. 2A). Then, we investigated the proliferation ability of CRC cells using CCK-8 assay. Consistent with the CBS overexpressing results, GYY4137 treatment inhibited the growth of HCT-116 (1.0 ~ 6.0 mM) and HT-29 cells (0.1 ~ 6.0 mM) in a dose-dependent manner (vs. 0 mM GYY4137, *p < 0.05, ***p < 0.001; Fig. 2B). In addition, results from 18-h CCK-8 and transwell assay demonstrated that exogenous H 2 S from GYY4137 attenuated the migration capacity of HCT-116 (1.0 ~ 6.0 mM) and HT-29 cells (0.1 ~ 6.0 mM) dose-dependently (**p < 0.01, ***p < 0.001; Fig. 2C-E). Taken together, our results show that CBS overexpression and the consequent over-production of H 2 S lead to an impaired proliferation and migration ability of CRC cells.

Overexpression of CBS attenuates CRC cell growth and liver metastasis in vivo.
To clarify the in vivo effect of CBS overexpression on CRC cell proliferation, we established xenograft mouse model by injecting HT-29 cells with stable CBS overexpression or control cells into the left dorsal flank of nude mice (n = 6 for each group). The results indicated that CBS overexpression inhibited tumor growth rate and the difference was significant on day 31 and 34 (*p < 0.05; Fig. 3A, B). We also performed IHC staining of harvested xenografts and the results showed that CD44 and Ki-67 expression level were significantly higher in the control group, which was quantified by H-Score (***p < 0.0001; Fig. 3C, D). Next, we assessed the effect of CBS overexpression on tumor metastasis by intrasplenic injection of HT-29 cells into nude mice (n = 6 for each group). Two out of 12 mice developed evident subcutaneous masses and was therefore excluded from further analysis. Eight weeks after injection, the CBS-overexpressing group developed significantly fewer and smaller liver metastasis nodules in gross morphology (*p < 0.05; Fig. 3E, F). Liver metastases from both groups were then confirmed by H&E staining and typical fields under light microscope were demonstrated (Fig. 3G). Together, these results demonstrate that CBS overexpression attenuates CRC cell growth and liver metastasis in vivo.

CD44 and the transcription factor SP-1 is involved in the inhibitory effect of CBS/H 2 S axis on CRC cells
CD44 is a large family of transmembrane glycoproteins well known for its pivotal role in regulating cell proliferation, migration, invasion, and stemness. To explore how CBS/H2S axis exerts its inhibitory effect on CRC cells, we examined the effect of CBS overexpression on stemness marker CD44. Flow cytometry analysis showed that, the MFI ratio of CD44 was significantly higher in the control group comparing to the CBS overexpressing group (Fig. 4A). Next, we determined the association between CBS overexpression and CD44 expression through RT-qPCR and western blotting in HT-29 cell lines and xenografts from nude mice model. The results suggested that CBS overexpression led to a significant reduction in CD44 variant (CD44v) expression on both mRNA and protein levels (Fig. 4B, C).
We then verified our findings with exogenous H 2 S donation from GYY4137 on HT-29 cell line. When given at a higher concentration (1.0 ~ 6.0 mM), a significant decrease in CD44v expression was observed in a dose-dependent manner (Fig. 4D). To further investigate whether the inhibitory effect of CBS/H 2 S on CD44v expression was dependent on a relatively high CBS level, we established a sh-CBS Caco-2 cell line by shRNA lentivirus system and a KO-CBS HT-29 cell line by CRISPR/ Cas9 technology (Fig. 4E). Western blotting results clearly demonstrated that CBS downregulation promoted CD44v expression (Fig. 4E).
Then, we set out to investigate the mechanism underlying the inhibitory effect of CBS/H 2 S on CD44v levels. It has been reported that the CD44 gene promoter region contains SP-1 binding sites [25], and that SP-1 phosphorylation level is associated with H 2 S and p38/MAPK pathways [26]. Therefore, we hypothesized that the participation of CD44v in the regulation of CBS/H 2 S axis on CRC cells was mediated through RNA binding protein, SP-1. We conducted ChIP assays followed by quantitative PCR with primers specifically targeting the SP-1 binding region in the promoter sequence of human CD44. The ChIP results showed that overexpression of CBS led to a significant decrease in SP-1 recruitment to the CD44 promoter (Fig. 4F). We then carried out an immunofluorescence assay to further determine the cellular distribution of SP-1. Semi-quantitive analysis showed a significant reduction in the colocalization of SP-1 and DAPI in CBSoverexpressing cells determined by Pearson's correlation coefficient, indicating a decreased nuclear enrichment of SP-1 in HT-29 cells with CBS overexpression (**p < 0.01, Fig. 4G, H).

Discussion
The potential role of H 2 S in colorectal cancer has been a subject of extensive studies. Controversy still exists concerning whether this rather new gasotransmitter exerts a pro-cancer or anti-cancer effect. IHC and western blotting analysis using human colon cancer specimens revealed a distinct increased expression of CBS in tumor tissues compared with adjacent normal mucosa slices. Contrary to the result at protein level from limited patient samples, no such difference could be detected on mRNA levels based on the analysis of GEPIA2 database, which contains global gene expression data from TCGA and GTEx (Additional file 1: Fig S1; http:// gepia2. cancerpku. cn/# index). Although previous studies have mainly focused on the pro-tumor aspect of CBS/H 2 S, including maintaining cellular bioenergetics, promoting tumorigenesis, and stimulating angiogenesis and vasorelaxation  [27], evidence from both in vitro and in vivo experiments supporting the anti-cancer effect of H 2 S are also accumulating [28].
To decipher the bewildering biological and pharmaceutical role of CBS/H 2 S axis, we conducted several in vitro and in vivo experiments, mainly focusing on cell lines with high CBS expression (HT-29 and HCT-116) [12]. The current study indicated that overexpression of CBS inhibited CRC cell proliferation, clone formation, sphere formation and migration. Consistent with the in vitro results, attenuated xenograft growth rate and reduced liver metastasis was observed in cells with CBS overexpression in nude mice model. We then identified the involvement of CD44 in the effect of CBS/H 2 S axis on CRC cells, which is a well-known transmembrane marker for its pro-cancer and stemness-maintenance function. It is worth noticing that the predominant form of CD44 examined in HT-29 cell line by western blotting was CD44v. Certain CD44 isoforms, such as CD44v6, are suggested to possess cancer-initiating ability [29]. CD44v was derived from mRNA alternative splicing and they were reported to have a positive correlation with the degree of tumor aggressiveness, especially the characteristics related to tumor metastasis [17][18][19][20][21] .
After verification of the CD44-mediated regulation pattern, we further explored the molecule mechanism underlying the regulatory effect of CBS/H 2 S on the expression of CD44. H 2 S has been reported to inhibit the phosphorylation process of a transcription factor, SP-1, and thus suppress its functional activity [26]. Moreover, SP-1 could bind to the promoter region of CD44 [25]. Thus, we conducted further experiments focusing on the function and nuclear translocation of SP-1. In line with our hypothesis, the results from ChIP assays indicated an attenuated interaction between CD44 mRNA and SP-1 in CBS-overexpressing HT-29 cell line. Finally, we discovered that overexpression of CBS inhibited nuclear enrichment of SP-1 through immunofluorescence staining and Pearson's correlation analysis. Taken together, our study suggested a SP-1/CD44-mediated inhibitory effect of CBS/H 2 S axis on the proliferation, migration and metastasis capacity of CRC cells.
The seemingly unorthodox results from our study could partly be explained by the dual-effect of H2S. Indeed, it has been reported that H 2 S, along with NO and CO, exhibited a pro-cancer effect at rather low concentrations and an anti-cancer action at higher concentrations [30]. Moreover, the tumor-suppressing role of different H 2 S donors has been preliminarily tested both in vitro and in vivo. Lee et al. first reported the anti-cancer effect of the slow-releasing H 2 S donor, GYY4137, by promoting cell cycle arrest and apoptosis [31]. Faris et al. discovered that exogenous administration of H 2 S suppresses proliferation in primary cultures of metastatic CRC cells by inducing an increase in intracellular flux of Ca 2+ [32]. Modis et al. found that an allosteric CBS activator, S-adenosyl-l-methionine (SAM), inhibited HCT-116 cell proliferation and bioenergetics at higher levels (3 mM) or after longer-term exposure (72 h), although the inhibitory effect appeared to result from CBS-independent pharmacological mechanisms [33]. Other potential mechanisms underlying the anti-cancer effect of H 2 S administration including uncontrolled cellular acidification and inhibition of cell survival signaling pathways [13].
As for the underlying mechanism, the participation of SP-1/CD44 in the regulation of CBS/H 2 S on CRC cells should be interpreted rather cautiously, bearing in mind the complexity of the signaling pathway and cascades triggered by the fluctuation in CBS expression and H 2 S level in different tissues. Our results showed that overexpression of CBS attenuated the recruitment of SP-1 to the promoter region of CD44 mRNA, suggesting that CBS/H 2 S potentially modulates CD44 expression via the transcription factor SP-1. Indeed, Wu et al. reported that overexpression of CSE, another important H 2 S synthase, significantly suppressed SP-1, p38 and ERK1/2 activation in rheumatoid arthritis, and that SP-1 activation was inhibited by p38 and ERK [26]. These data suggested that H 2 S might negatively regulate SP-1 activation through inhibition of MAPK pathway. H 2 S modulated protein activity mainly through two ways: protein sulfhydration or intracellular formation of polysulfides followed by oxidative inactivation of proteins [34]. The exact mechanisms of how CBS/H 2 S axis controls CD44 expression via SP-1, either via protein sulfhydration or oxidative stress, remain to be further explored.
Based on the results from our study as well as many others, we hypothesized that, at a relatively low expression level, the upregulation of CBS/H 2 S triggers a series of downstream reactions that promote the growth and dissemination of CRC cells, which override the tumorsuppressing effect stemming from the downregulation of CD44. When the CBS/H 2 S axis is already highly-activated, however, a further upregulation of CBS or exogenous donation of H 2 S would lead to a major decrease in CD44 expression. Under such circumstances, CBS overexpression and exogenous H 2 S manifests tumorsuppressing activities in CRC cells, including inhibition of proliferation, decreased clone formation, attenuation of migration, as well as an impaired capacity of in vivo xenograft growth and liver metastasis. Apart from colon cancer, the anti-tumor activity of H 2 S has been widely discovered in various cancer cell types, including gastric, hepatic, breast and melanoma cancer cells [35]. Although clinical trials investigating H 2 S donor for cancer therapy have not yet been conducted, given the context-dependent role of CBS/H 2 S in tumor development, the therapeutic potential of H 2 S supplement in a subgroup of patients with high levels of CBS expression merits additional study.

Conclusions
In conclusion, our study indicated that endogenous overexpression of CBS as well as exogenous H 2 S could inhibit CRC cell proliferation and migration both in vivo and in vitro. Moreover, the anti-tumor activity of CBS/H 2 S axis was largely attributed to the inhibition of a pivotal stem cell marker, CD44. CBS/H 2 S negatively modulated CD44 expression through the transcription factor SP-1. Further